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Analysis of Organophosphate Pesticides in the Blood Samples of Agricultural Workers of District Gujranwala, Punjab

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Analysis of Organophosphate Pesticides in the Blood Samples of Agricultural Workers of District Gujranwala, Punjab ANALYSIS OF ORGANOPHOSPHATE PESTICIDES IN THE BLOOD SAMPLES OF AGRICULTURAL WORKERS OF DISTRICT GUJRANWALA, PUNJAB. _____________________________________________________________________ SAFFORA RIAZ ________________________________________________________________________ DEPARTMENT OF ZOOLOGY LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2017 ANALYSIS OF ORGANOPHOSPHATE PESTICIDES IN THE BLOOD SAMPLES OF AGRICULTURAL WORKERS OF DISTRICT GUJRANWALA, PUNJAB. _____________________________________________________________________ A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY By SAFFORA RIAZ ________________________________________________________________________ DEPARTMENT OF ZOOLOGY LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2017 CERTIFICATE This is to certify that the research work described in thesis entitled “Analysis of organophosphate pesticides in the blood samples of agricultural workers of district Gujranwala, Punjab.” submitted by Ms. SAFFORA RIAZ to Department of Zoology, Lahore College for Women University has been carried out under our direct supervision. We have personally gone through the raw data and certify the correctness and authenticity of all results reported herein. We further certify that thesis data has not been used in part or full, in a manuscript already submitted or in the process of submission in partial fulfillment of the award of any other degree from any other institution or home or abroad. We also certify that the enclosed manuscript has been prepared under our supervision and we endorse its evaluation for the award of PhD degree through the official procedure of University. ________________ Supervisor Co Supervisor Prof. Dr. Farkhanda Manzoor Dr. Nasir Mahmood Head Department of Zoology Assistant Professor LCWU, Lahore Department of Biochemistry; Department of Date: Human Genetics and Molecular Biology University of Health Sciences, Lahore, Pakistan. Date: Verified By Chairman ________________ Prof. Dr. Farkhanda Manzoor Head Department of Zoology Stamp: _________________ Controller of Examination Stamp Date: ___________ DEDICATION I WOULD LIKE TO DEDICATE ALL OF MY EFFEORTS TO MY FATHER RIAZ AHMED CHATTHA WHO SUPPORTS ME FINANCIALLY, MORALLY AND ENCOURGE ME TO EVERY STEP OF MY LIFE AND TO MY MOTHER GHULAM ZOHRA FOR HER PATIENCE ACKNOWLEDGEMENT The whole praise to ALLAH Almighty the sovereign Power, the creator of the universe, who gave us knowledge, with which we are trying to understand the laws of nature and conquer the universe; who made me the super creature; blessed me with knowledge and enabled me to accomplish this task. I offer my humblest and sincere words of thanks to the Holy Prophet Muhammad (PBUH), who is forever a torch of guidance for humanity. I feel all words on the earth just failing to express my deep sense of gratitude to our respected Vice Chancelor, Prof. Dr. Rukhsana Kausar for giving me opportunities to complete my research thesis of Ph.D Zoology. I respectfully express my deepest appreciation to our respected, distinguished and affectionate director research Prof. Dr. Shagufta Naz for providing opportunities to complete my research thesis. I am also obliged to my supervisor Prof. Dr. Farkhanda Manzooor, Head of Zoology Department, Lahore College Women University, Lahore for her sincerest guidance, critical suggestions. She has been so kind and supportive with her patience and knowledge and a source of inspiration for me. I respectfully express my deepest appreciation and acknowledgement to Dr. Nasir Mahmood, Assistant Professor, Department of Biochemistry; Department of Human Genetics and Molecular Biology, University of Health Sciences, Lahore, Pakistan for his keen interest, providing technical guidance, research facilities and valuable suggestions. I am highly thankful to my sister Sadia Tabassum and my brothers Sami Ullah and Dr. Ali for encourage me to achieve this goal. I am also greatly indebted to my friends Saadiya Asma, Ayisha butt, Touseef Kazmi, Hafsa Memoona and Tehziba Kausar and my lab fellows for their friendly attitude, support and sympathetic encouragements. I also want to pay thanks to laboratory staff of Zoology Department of LCWU for their cooperation. SAFFORA RIAZ CONTENTS Title Page No. List of Tables i List of Figures ii List of Abbreviations v Abstract viii Chapter 1 : Introduction 1 Chapter 2: Review of Literature 14 Chapter 3: Materials and Methods 48 3.1: Study design 48 3.2: Questionnaire based survey 50 3.3: Sample size 50 3.4:Sample collection and processing 50 3.5: Hematological analysis 50 3.6: Serological analysis 51 3.6.1: Alanine aminotransferase (ALT) 51 3.6.1.1: Procedure 51 3.6.2: Aspartate aminotransferase (AST) 52 3.6.2.1: Procedure 52 3.7. Biochemical analysis 53 3.7.1: Determination of cholinesterase activity 53 3.7.2: Acetylcholine Assay 53 3.8:Determination of levels of immunoglobulins 54 3.8.1:Protocol 54 3.9: High Performance Liquid Chromatography 55 3.9.1: Preparation of Standard of pesticides 55 3.9.2: Preparation of the serum sample 55 3.9.2.1: Protein Precipitation 55 3.9.2.2: Filtration assembly 56 3.9.2.3: Sonication 56 3.9.3: Optimization of condition for HPLC 56 3.9.4: Quntification of pesticides by HPLC 62 3.9.4.1: Area Height Method 62 3.10: Tuberculosis detection in suptum of spray workers 62 3.10.1: Molecular detection of tuberculosis 62 3.10.2: Genomic DNA isolation 63 3.10.3: Agarose gel electrophoresis of genomic DNA 64 3.10.3.1: Quantification of DNA 64 3.10.4: Preparation of 1% the agarose gel for PCR product 64 Analysis 3.10.4.1: Loading of the gel 65 3.10.5: Polymerase chain reaction 65 3.10.6: Molecular differentiation of M. tuberculosis and M. 68 Bovis 3.10.6.1: Gene clean of amplified DNA fragment for 69 sequencing 3.10.6.2: Sequencing of selected samples 69 3.11: Statistical analysis 69 Chapter 4: Results 70 4.1: Demographic features of agricultural spray workers of district 70 Gujranwala. 4.2: Educational level of workers by gender 71 4.3: Awareness and practices of pesticides use by gender 72 4.4: Personal equipment and clothing while spraying 73 4.5: Pesticides related acute health problems 75 4.6: Hematological profiles of agricultural spray workers 77 4.6.1: Complete blood profile of male agricultural spray 77 workers and non spray workers: 4.6.2: Complete blood profile of female agricultural spray 78 workers and female non spray workers. 4.7: Analysis of serological parameters of agriculture spray 80 workers 4.7.1: Liver enzymes ALT and AST analysis of male 80 agriculture spray workers in comparison to male non spray workers 4.7.2: Liver enzymes ALT and AST analysis of female 80 agriculture spray workers in comparison to female non spray workers: 4.8: Acetylcholinestrase standardization 82 4.9. Biochemical analysis of acetylcholinestrase 86 4.9.1. Analysis of level of acetylcholinestrase 86 concentration in blood serum of male spray workers 4.9.2. Analysis of level of acetylcholinestrase concentration in 87 blood serum of female spray workers 4.10: Immunoglobulin concentration in blood serum of male 88 spray workers 4.10.2: Level of immunoglobulin IgA 90 4.10.3: Level of immunoglobulin IgM 91 4.10.4: Levels of immunoglobulin IgE 92 4.11: Standaridization of HPLC 93 4.12: Pesticide residues detection and quantification blood 122 serum of spray workers 4.12.1: Descriptive statistical data of pesticides 123 quantification in serum samples of spray workers 4.13: Detection of different species of mycobacteria 124 4.13.1: DNA isolation and quantification 124 4.13.2: Molecular differentiation of M. tuberculosis 125 and M. bovis PCR with the use of primer set N & S. 4.13.3: Molecular detection of M. tuberculosis and M. 126 bovis in pesticides exposed workers. 4.13.4: Sequencing of targeted hup B gene 127 Chapter 5: Discussion 133 References 143 Annexures x Plagiarism Report xxv List of Publications and reprints xxvii i List of Tables Table No. Title Page No. 3.9.3.1: HPLC conditions for the pesticides analysis in serum. 56 3.9.3.2: Optimized HPLC conditions for dichlorovos pesticide in blood serum of 57 agricultural workers. 3.9.3.3: Optimized HPLC conditions for profenophos pesticides in blood serum 58 of agricultural workers. 3.9.3.4: Optimized HPLC conditions for chlorpyrifos pesticides in blood serum 59 of agricultural workers. 3.9.3.5: Optimized HPLC conditions for malathione pesticides in blood serum of 60 agricultural workers. 3.9.3.6: Optimized HPLC conditions for imidacloprid pesticides in blood serum 61 of agricultural workers. 3.10.5.1. Concentration and volume of reagents used for PCR 66 4.6.1: Complete blood profile of male agricultural spray workers and non - 77 spray workers. 4.6.2: Complete blood profile of female agricultural spray workers and 78 corresponding female control subjects. 4.7.1: Comparison between liver enzymes ALT and AST in male agricultural 80 spray operators and corresponding controls. 4.7.2: Comparison between liver enzymes ALT and AST in female 80 agricultural spray operators and corresponding controls. 4.9.1: Acetylcholinestrase concentration level in male spray workers and their 86 corresponding control subjects. 4.9.2: Acetylcholinestrase concentration level in female spray workers and 87 their corresponding control subjects 4.10.1.1: Levels of immunoglobulin in blood serum of males agricultural spray 88 workers of exposed to pesticides as compared to non spray workers of district Gujranwala. 4.10.1.2: Levels of immunoglobulin in blood serum of females agricultural spray 89 workers of exposed to pesticides as compared to non spray workers of district Gujranwala. 4.12: Number of spray workers with positive concentration of pesticides. 122 Descriptive statistical data of pesticides residues detected in serum 4.12.1: samples of female spray workers. 123 4.12.2: Descriptive statistical data of pesticides residues detected in serum 123 samples of female spray workers.
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